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Received: 18 March 2019    Revised: 26 March 2019    Accepted: 26 March 2019

DOI: 10.1111/dgd.12608

REVIEW ARTICLE

Nanopore sequencing: Review of potential applications in

functional genomics

Nobuaki Kono  | Kazuharu Arakawa

Institute for Advanced Biosciences, Keio

University, Tsuruoka, Yamagata, Japan
Correspondence
Kazuharu Arakawa, Institute for Advanced
Biosciences, Keio University, Tsuruoka,
Yamagata, Japan.
Email: gaou@sfc.keio.ac.jp
Funding information
ImPACT Program of Council for Science,
Technology and Innovation (Cabinet Office,
Government of Japan); Yamagata Prefectural
Government and Tsuruoka City, Japan
Abstract
Molecular biology has been led by various measurement technologies, and increased
throughput has developed omics analysis. The development of massively parallel se‐
quencing technology has enabled access to fundamental molecular data and revealed
genomic and transcriptomic signatures. Nanopore sequencers have driven such evo‐
lution to the next stage. Oxford Nanopore Technologies Inc. provides a new type of
single molecule sequencer using protein nanopore that realizes direct sequencing
without DNA synthesizing or amplification. This nanopore sequencer can sequence
an ultra‐long read limited by the input nucleotide length, or can determine DNA/RNA
modifications. Recently, many fields such as medicine, epidemiology, ecology, and
education have benefited from this technology. In this review, we explain the fea‐
tures and functions of the nanopore sequencer, introduce various situations where it
has been used as a critical technology, and expected future applications.

KEYWORDS
long reads, nanopore sequencing, next generation sequencing

1 | I NTRO D U C TI O N
Novel technologies that visualize the unseen or detect the unde‐
tectable have always contributed to breakthroughs in scientific dis‐
coveries, and the rapid advent of high‐throughput and affordable
DNA sequencing technologies has undoubtedly been the key driving
force in the progress of life sciences over the last decade (Goodwin,
Mcpherson, & Mccombie, 2016). Genomic information has also been
one of the cores of molecular biology, providing and assisting tools
to probe the genome, its structure, epigenetics, gene expression,
and a multitude of other applications. Latest in the line of DNA
sequencers are the nanopore sequencers (Heather & Chain, 2016;
Jain, Olsen, Paten, & Akeson, 2016; Mardis, 2013), successfully
commercialized by Oxford Nanopore Technologies (ONT) (Brown &
Clarke, 2016). While a variety of sequencing approaches exist such
as pyrosequencing, sequencing by synthesis, sequencing by ligation,
and single molecule real time (SMRT) sequencing, DNA sequencing
methods since Sanger sequencing predominantly relied on the pro‐
cess of DNA synthesis (Clarke et al., 2009; Eid et al., 2009; Schadt,
Turner, & Kasarskis, 2010). Nanopore sequencing distinguishes itself
from these previous approaches, in that it directly detects the nu‐
cleotides without active DNA synthesis, as a long stretch of single
stranded DNA passes through a protein nanopore that is stabilized
in an electrically resistant polymer membrane (Branton et al., 2008;
Feng, Zhang, Ying, Wang, & Du, 2015). By setting a voltage across
this membrane, sensors detect the ionic current changes shifted by
nucleotides occupying the pore in real time as the DNA molecule
passes through. History of the development of nanopore sequenc‐
ing of DNA, as well as detailed methods for sequencing, is already
reviewed in depth elsewhere (review in (Deamer, Akeson, & Branton,
2016)) and is beyond the scope of this review. Here, we focus on the
current advantages as well as the limitations of the ONT nanopore
sequencer, reviewing research publications available thus far. It
should be noted, however, that in a rapidly evolving technology like
nanopore sequencing, reviewing efforts can also quickly become
outdated.
Nanopore's unique sequencing method provides multiple ad‐
vantages over existing technologies. Firstly, nanopore sequencing

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does not require imaging equipment to detect the nucleotides,
allowing the system to scale down in size to a portable level. The
current MinION Mk1B device only weighs 90 g, measuring only
3.3 × 10.5 × 2.3 cm in size, fitting in the palm of one's hand. The
cost of the device is also much lower compared to other massively
parallel sequencers, the initial cost being only around $1,000, in‐
cluding the device and initial set of reagents. MinION devices can
be powered through the USB (Universal Serial Bus) port of laptop
computers, such that sequencing can be conducted anywhere, even
in the field. Lack of an image analysis step also allows real time base
calling during sequencing, realizing rapid detection of target DNA
for the screening of pathogens from clinical samples, for example.
Secondly, since nanopore sequencing directly detects the input mol‐
ecule without DNA amplification or synthesis, there is no apparent
limit to the length of DNA that can be sequenced. The challenge
in read length using nanopore sequencers therefore is not in the
sequencing technology itself, but in the library preparation step,
which needs to extract and load intact extremely high molecular
weight (HMW) DNA into the flow cell of the sequencer. Sequenced
reads exceeding a mega‐base have been reported, demonstrating
the extraordinary capabilities of the nanopore device in sequenc‐
ing extremely long stretches of the DNA molecule (Jain et al., 2018).
The extreme long reads enable de novo genome assembly without
preparation of complex mate‐pair libraries typically required with
short read sequencing. The extremely long reads are also useful
to determine sequences of genomic region containing long repeti‐
tive sequences, which is difficult with short read sequencing. Long
reads also allow the study of structural variations within the genome
(Cretu Stancu et al., 2017). Moreover, detection of the ionic current
changes shifted by the nucleotides passing through the nanopore is
not limited to the canonical four nucleobases of adenine, guanine,
cytosine and thymine. Taking advantage of the nature of unamplified
direct sequencing, nanopore sequencing can directly observe base
modifications such as methylations (Rand et al., 2017; Simpson et al.,
2017), and even direct sequencing of RNA molecules containing ura‐
cil bases (Garalde et al., 2018).
The main current drawback of nanopore sequencing, as with
other long read sequencers, is the relatively high error rate com‐
pared to short read sequencing. Current error rates range from 5%
to 20%, dependent on the type of molecules and library prepara‐
tion methods, and the errors include both insertions and deletions
(Rang, Kloosterman, & De Ridder, 2018). Unlike SMRT sequencing of
PacBio, there seems to be systematic error in nanopore sequencing
(Jain et al., 2018), so error correction typically requires additional
short read sequence data. Data yield also varies rather significantly
depending on the input, which is also difficult to predict. On the
other hand, active developments in bioinformatics for base‐calling
and error correction, as well as optimization on library preparation
steps and new pore developments by ONT, are rapidly improving
on these issues. Throughput of the system is greatly enhanced;
for example, by the recent introduction of the PromethION sys‐
tem, where a single flow cell can yield 50–100 Gbp (typical yield of
MinION system is 5–10 Gbp) and 24 flow cells can be run in parallel.
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Improvements in base‐caller software from Hidden Markov Model
(HMM) based methods to Recurrent Neural Network (RNN) based
algorithms enhanced base‐level accuracy by 2%–5% (Rang et al.,
2018; Teng et al., 2018). In the following sections, we review each of
the above points in detail.
2 | U LTR A‐ LO N G R E A DS ‐ “ W H A LE
WATC H I N G ”
The official library preparation protocol with adapter ligation pro‐
vided by ONT recommends an optional fragmentation step for the
input DNA to average in the 8 kbp range, in order to have optimal
molar concentration of adaptor‐ligated DNA ends to meet the na‐
nopores during sequencing to gain maximal throughput, which is
around 0.2 pM. However, since there is no apparent technical limit
for the size of DNA to be sequenced, the nanopore community
primarily led by Loman et al. called out for “whale spotting” with
nanopore (http://lab.loman.net/2017/03/09/ultrareads-for-nanop‐
ore/). A “whale” is a comparative measurement system that relates
ultra‐long DNA bp to the nearly equivalent numbers of grams: for
example, one of the smallest whales is the narwhal, weighing around
940 kg, and read lengths of 940 kbp to 1 mbp were the initial chal‐
lenge. In order to load such HMW DNA into the sequencer, every
step of DNA handling required reconsideration. Typical DNA extrac‐
tion methods using commercial kits such as the silica spin column
usually result in relatively shorter DNA molecules, typically below
50 kbp. Drying of DNA to eliminate residual ethanol during purifi‐
cation, clean‐up and concentration with paramagnetic Solid Phase
Reversible Immobilization (SPRI) beads, and even the pipetting
of DNA solution result in fragmentation of ultra‐long DNA mol‐
ecules. Therefore, Quick developed a protocol based on the classic
Sambrook method (Sambrook & Russell, 2001) of using phenol‐chlo‐
roform‐isoamyl alcohol with wide‐bore pipette tips to extract HMW
DNA from cultured cells, and then prepared a nanopore library with
a Rapid Sequencing Kit (ONT) that utilized transposons to add the
sequencing adapters with minimal pipetting steps (Quick J. 2018.
Ultra‐long read sequencing protocol for RAD004. https://www.
protocols.io/view/ultra-long-read-sequencing-protocol-for-rad004-
mrxc57n). A larger amount of input DNA was also required due to
the decreased molarity of DNA ends with ultra‐long molecules. One
of the very first “whales” was successfully spotted with this protocol
for human culture cells, where an ultra‐long read of length 882 kbp
was mapped to the reference spanning a 950 kbp region (Jain et al.,
2018). With a sequencing speed of 450 bp/s for 1D kits using R9.4
flow cells, the “whale watching” of this single read took more than
30 min as it passed through the pore. Bioinformatics methods were
also required to observe the entirety of such ultra‐long sequencing,
for the software controlling the sequencing (MinKNOW) errone‐
ously subdivided some of the continuous data streams of ultra‐long
reads into shorter multiple reads. A software to observe such dis‐
continuity, designated BulkVis, was developed, which observed
the largest “whale” yet at 2,272,580 bp (Payne, Holmes, Rakyan, &
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318       KONO and ARAKAWA
Loose, 2018). This single ultra‐long read is comparable to the size
of smaller bacterial chromosomes such as those of Bifidobacterium
longum NCC2705, with 2,256,640 bp (Schell et al., 2002), or nearly
one half of the Escherichia coli genome, demonstrating the potential
to sequence chromosome‐level reads with nanopore technology.
It is also interesting that this kind of new technology reevaluates
the rather classic methods of DNA handling, such as the use of the
phenol‐chloroform‐isoamyl alcohol method, vacuum concentration,
pulse‐field gel electrophoresis (PFGE) with gel plugs and tapping or
gentle rotation as opposed to spin columns, the use of wide bore
pipette tips, pipette tip cutting, vortexing, SPRI clean‐up, or regular
submarine gel electrophoresis. In order to maximize the read length
in nanopore sequencing, one needs to take extra care for such HMW
DNA handling throughout the library preparation steps.
The extraction protocol should be adjusted according to the
target species, and cell lysis and homogenization procedures have
a profound influence on the extracted nucleotide quality for tissue
samples, unlike Gram‐negative bacteria or cultured cells that lack a
thick cell wall or exoskeleton. Therefore, homogenization and DNA
extraction methods should be optimized for each species, especially
for plants or arthropods that harbor thick cell walls and cuticular
exoskeletons. In the case of plant genomes such as the fastest‐grow‐
ing angiosperm (greater duckweed) or tropical timber trees (teak),
HMW genomic DNA extraction has been successfully performed
by a CTAB method (Hoang et al., 2018; Yasodha et al., 2018), fol‐
lowing homogenization steps to break the cell wall, where grinding
with liquid nitrogen is generally used to crush the cell wall while
cellular enzymes remain inactivated. After sufficient homogeniza‐
tion, samples are then resuspended in detergent‐based extraction
buffers containing cetyltrimethylammonium bromide (CTAB). Due
to the abundance of polysaccharides in plants, the CTAB method
is established as the best detergent for purifying DNA from plant
material (Murray & Thompson, 1980). Likewise, in insects such as
harlequin ladybird or fruit fly, a dissected tissue or a whole specimen
frozen with liquid nitrogen is ground using a pestle, and HMW DNA
extraction is subsequently performed with Genomic‐tip 500/G kit
(QIAGEN) or Blood & Cell Culture kit (QIAGEN) (Gautier et al., 2018;
Miller, Staber, Zeitlinger, & Hawley, 2018). For vertebrates such as
clownfish or eel, suitable tissue samples such as muscles or livers are
selected (Jansen et al., 2017; Tan et al., 2018).
Coupled with the portable, real time, single molecule nature of
the nanopore sequencing technology, it is worth noting that the
long reads are expected to contribute to translational applications
in genomic medicine and clinical testing (Ameur, Kloosterman, &
Hestand, 2019). Notably, long reads are critical in structural vari‐
ation analysis and the complete sequencing of repetitive DNA
contents of clinical utility. Norris and colleagues demonstrated
that nanopore sequencers could detect large‐scale structural vari‐
ations, including large deletions, inversions, and translocations re‐
lated to the inactivation of tumor suppressor genes in pancreatic
cancer, with very few reads (Norris, Workman, Fan, Eshleman, &
Timp, 2016). Another group developed a new bioinformatics tool,
NanoSV, for structural variation detection with nanopore data
using split read mapping (Cretu Stancu et al., 2017). Mitsuhashi
and colleagues successfully identified a full‐length microsatellite
repeat spanning 49,877 bp in the D4Z4 array responsible for fa‐
cioscapulohumeral muscular dystrophy (Mitsuhashi, Nakagawa,
et al., 2017). In the case of HLA genotyping, the latest MinION
accuracy has reached a quality that offers cost‐effective and
scalable genotyping with minor shortcomings (Lang et al., 2018).
Furthermore, hybrid assembly of combined short‐ and long‐reads
could resolve the structure and chromosomal insertion site of
the antibiotic resistance island in Salmonella Typhi Haplotype 58
(Ashton et al., 2015), opening up numerous possibilities for real‐
time genomic epidemiology.
3 | LO N G R E A D A S S E M B LY S TR ATEG I E S
In short read assembly, the de Bruijn graph (DBG) algorithm has
been most commonly used due to its accuracy and speed (Lu,
Giordano, & Ning, 2016). The DBG algorithm splits sequenced
reads into their k‐mer components and constructs a graph with
connecting pairs of k‐mers based on whether they have k‐1 com‐
mon nucleotides. This approach, however, is highly dependent
on the determination of the k‐mer graph, which is not suitable
for long reads with innately high error rates, and cannot fully
take advantage of the longer lengths of the reads, to take ac‐
count of complex genomic features including structural variation
or non‐random elements (Sohn & Nam, 2018). Long read assem‐
bly therefore revived interest in an overlap‐layout‐consensus
(OLC) algorithm with all‐against‐all pairwise alignment, which
is flexible in read length and robust to error, allowing for a de‐
crease in chimeric contigs or assembly bubbles. Canu has been
the most popular assembly software used in numerous nanop‐
ore assembly projects, which includes tf‐idf weighted MinHas
for fast and accurate adaptive overlapping Nanopore‐only error
correction and using the raw electric current signal information
can significantly improve base‐level accuracies to around 99%,
as demonstrated in the whole genome assembly of E. coli K‐12
MG1655 (Loman, Quick, & Simpson, 2015). This approach, how‐
ever, still ends with a higher base‐level error rate in comparison
to short read assembly, and is highly computationally intensive.
A hybrid strategy utilizing short Illumina reads is therefore com‐
monly adopted to polish the assembly (Figure 1a) (Giordano
et al., 2017; Istace et al., 2017). Multiple rounds of polishing are
often necessary, where the error correction process is validated
using Benchmarking Universal Single‐Copy Orthologs (BUSCO)
completeness scores (Jain et al., 2018; Simao, Waterhouse,
Ioannidis, Kriventseva, & Zdobnov, 2015; Tan et al., 2018; Tyson
et al., 2018). Several assemblers incorporate the hybrid error
correction step prior to the assembly, in place of the initial over‐
lap‐based error correction (Bankevich et al., 2012; Zimin et al.,
2013). While hybrid error correction is computationally efficient
and generally results in highly accurate sequences, it should
be noted that the disadvantage of short reads remains in this
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F I G U R E 1   (Long read assembly) The nanopore read assembly requires error correction, assembly, and polishing processes. Error
correction can be performed by hybrid or long read only approaches. Hybrid methods use high‐accuracy short read. Assembled contigs
are polished to improve the consensus sequence by using high‐accuracy read or raw current data obtained by nanopore sequencer. (Splice
variant analysis) Long read obtained by direct sequencing allows detection of structural variation as it is. Therefore, the splice variant
analysis does not require assembly

approach, where error correction of non‐random or repetitive
regions is difficult.
4 | TR A N S C R I P TO M E A N A LYS I S A N D
D I R EC T R N A S EQ U E N C I N G
The advantages of long reads are not limited to the study of genomic
DNA, but are also useful in the study of RNA sequences. It is
especially pertinent to uncover the diversity of alternative splicing
isoforms and their expression levels, which are often difficult to de‐
lineate with short reads (Figure 1b). Consequently, the combination
of long and short reads is reported to increase transcriptome analy‐
sis performance (Weirather et al., 2017). Use of long reads for the
analysis of splice isoforms has clear advantages over short reads, in
that there is no need for read mapping or assembly to figure out
the isoform structures, but one can instead simply look at the en‐
tire sequence to pinpoint the isoform. The Dscam1 (Down syndrome
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320       KONO and ARAKAWA
cell adhesion molecule 1, (Schmucker et al., 2000)) gene structure
in Drosophila, which produces a total of 38,016 different isoforms
via alternative splicing arising from four variable exon clusters, is re‐
garded as “the most complicated alternatively spliced gene known in
nature” (Bolisetty, Rajadinakaran, & Graveley, 2015). Nanopore full‐
length cDNA sequencing analysis, however, successfully identified
7,899 full‐length isoforms of this gene. The accurate identification
of incorporated exon structures using long reads is also powerful in
its application to evidence‐based gene structural annotation. Cook
and colleagues developed an automated gene structure annota‐
tion pipeline named LoReAn (long‐read annotation), in which they
demonstrated that a full‐length cDNA sequence could assist correct
annotation of the gene structures in Arabidopsis thaliana and Oryza
sativa (Cook et al., 2019).
Long read cDNA sequencing is also possible with other plat‐
forms, but the nanopore sequencer is the only device that has made
the direct sequencing of long stretches of RNA molecule possible
(Garalde et al., 2018). Although tRNA detection by nanopore has pre‐
viously been explored and reported (Henley et al., 2016; Smith, Abu‐
Shumays, Akeson, & Bernick, 2015), the ONT nanopore sequencer
commercialized direct RNA sequencing kits in 2017. Conventional
“RNA‐seq” with short reads, which is rather inappropriately termed
so since the RNA molecules are not directly sequenced (Hrdlickova,
Toloue, & Tian, 2017), requires reverse transcription (RT) of the tem‐
plate RNA molecule into complementary DNA (cDNA), which is typ‐
ically further amplified by PCR. Direct RNA sequencing (again, it is
unfortunate that true RNA sequencing has to add the word “direct”
to differentiate itself from conventional “RNA‐Seq”), on the other
hand, directly detects the ribonucleobases passing through the pore
without RT or PCR amplification, and this approach is free of pos‐
sible biases or misamplifications introduced during such steps. The
gene annotation process is simplified by the direct RNA sequenc‐
ing, and it has therefore allowed the identification of more com‐
plex or novel transcript isoforms genome‐wide (Byrne et al., 2017;
Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differ‐
entiate transcript haplotypes as well as to identify 3′ poly (A) tail
lengths (Workman et al., 2018). Such an application is also practical
for the study of RNA viral genomes, because the genome has multi‐
ple reading frames, anti‐sense gene locations, inefficient termination
signals, and complex splice forms, and gene annotation is challenging
using the conventional RNA‐seq method (Depledge et al., 2018).
Moreover, another strength of unamplified or non‐reverse‐tran‐
scribed direct RNA sequencing is the detection of nucleotide modifi‐
cations. Since the presence of modified bases results in altered ionic
current signal from the unmodified base as it passes through the
pore, nanopore sequencing is able to detect the modifications with‐
out any additional sample preparation. Rand and colleagues demon‐
strated that three cytosine variants within DNA molecules could be
detected with an accuracy of 80% using an HMM‐HDP (HMM with
a Hierarchical Dirichlet Process) (Rand et al., 2017), and Simpson and
colleagues also proved that the nanopore sequencer could detect
the 5‐methylcytosine (5‐mC) at up to 95% accuracy using HMM
(Simpson et al., 2017). For RNA modification, N6‐methyladenosine
(m6A) or 5‐mC is the most common internal modifications in mRNA
that are implicated in various RNA metabolism and regulations, and
nanopore sequencing can identify these modifications (Garalde
et al., 2018). A comprehensive study of the human poly (A) tran‐
scriptome using direct RNA sequencing has recently been reported,
including the detection of these modifications as well as A‐to‐I RNA
editing (Workman et al., 2018). Although it should be noted that the
error rate of direct RNA sequencing exceeds the already high error
rate of nanopore DNA sequencing, the ability to directly study RNA
molecules could open up a wide range of research that are otherwise
not possible.
5 | R E A L TI M E A N D P O RTA B LE
The excellent portability of a MinION device that can be bus‐pow‐
ered from a mobile PC can make a significant contribution in the
context of in‐the‐field sequencing (Ameur et al., 2019; Quick et al.,
2017), which has already been demonstrated in extreme places
such as an academic conference venue (Ii et al., 2019), coal fields
(Edwards et al., 2017), the arctic (Edwards, Debbonaire, Sattler,
Mur, & Hodson, 2016), Antarctica (Johnson, Zaikova, Goerlitz, Bai,
& Tighe, 2017), and even on the International Space Station (Castro‐
Wallace et al., 2017). For these purposes, mobile lab approaches for
DNA extraction and library preparation have been explored using
devices like VolTRAX V2 or Bento Lab (all‐in‐one DNA laboratory
including thermal cycler: https://www.bento.bio), and experimen‐
tal protocol developments are also underway, such as those using
LAMP (loop‐mediated isothermal amplification) methods instead of
conventional PCR to eliminate the need for a thermal cycler. LAMP
only requires a water bath to conduct its isothermal amplification,
and Imai and colleagues demonstrated an identification system for
human Plasmodium species (Imai et al., 2017). Yamagishi and col‐
leagues also reported the serotyping system for dengue virus in a
single day (Yamagishi et al., 2017) using this approach.
Moreover, the real time nature of the device, where DNA mol‐
ecules passing through the pore are immediately base‐called on a
per‐read basis as they are sequenced, becomes a more rapid and
highly robust alternative to conventional clinical testing such as che‐
miluminescence, ELISA, immune chromatographic tests (ICT), and
real‐time PCR. Greninger and colleagues implemented a web‐based
pipeline for real‐time analysis (MetaPORE) and made it possible to
detect Chikungunya virus, Ebola virus, and hepatitis C virus simul‐
taneously from a human blood sample with nanopore sequencing
(Greninger et al., 2015). Likewise, Mitsuhashi and colleagues de‐
veloped a real‐time identification of bacterial composition within
2 hr of obtaining a sample (Mitsuhashi, Kryukov, et al., 2017), and
Quick et al. (2015) showed that genotyping of a hospital outbreak of
Salmonella could be carried out in less than half a day. Furthermore,
combining the portable and real‐time nature of the MinION device,
it has been adopted for the surveillance and monitoring of viral out‐
breaks in resource‐limited locations. The Zika virus outbreak was
declared an international public health emergency by the World
KONO and ARAKAWA
Health Organization (WHO) in 2016, and the Zika in Brazil Real‐time
Analysis (ZiBRA) project was established to sequence a thousand ge‐
nomes from Brazil to monitor the epidemiological information (Faria
et al., 2016). Quick et al. (2017) thus developed a protocol to rapidly
obtain the complete genome of the Zika virus from clinical samples
using the MinION sequencer, encompassing sample preparation to
bioinformatics analysis. The same research group also developed a
genomic surveillance system using the nanopore sequencer for the
Ebola outbreak in West Africa (Quick et al., 2016). This system takes
as little as 15–60 min sequencing after receiving the blood sample,
realizing an on‐the‐spot virus testing without the need for the trans‐
portation of hazardous specimens. Furthermore, Runtuwene and
colleagues demonstrated a new MinION application for genotyping
analysis of the malaria parasite Plasmodium falciparum in Indonesia
(Runtuwene et al., 2018), stressing the potential of the nanopore ge‐
notyping approach as an effective and practical alternative for the
diagnosis of the malaria parasite.
6 | U S E C A S E: H I G H LY R E PE TITI V E
S PI D E R S I LK G E N E S
The improved accessibility of sequencing technology makes it easier
to access genomic data, and contributes significantly to molecular
biology in non‐classical model organisms (Russell et al., 2017). Long
read assemblies are especially useful in this regard in complex large
eukaryotic genomes, typically with numerous non‐random features
such as highly repetitive sequences and polyploidy (Kono, Nakamura,
Ohtoshi, Pedrazzoli Moran, et al., 2019; Kono, Nakamura, Ohtoshi,
Tomita, Numata, et al., 2019). As one example, we here introduce the
de novo sequencing of a spider silk protein (spidroin) gene. The spi‐
der silk exhibits extraordinary physio‐chemical properties for poten‐
tial industrial applications as a multifunctional protein material, such
as high toughness, high tensile strength per density, and thermosta‐
bility (Blamires, Blackledge, & Tso, 2017; Omenetto & Kaplan, 2010).
F I G U R E 2   The dot plots represent the nanopore reads covering full length gene. The area where dot concentrates indicates the
receptive region. Red and blue box mean N‐ and C‐terminus domain. Gray box means intron region. (a) and (b) show MiSp and AcSp gene
reads obtained by Araneus ventricosus genomic DNA. These dotplots were generated using LAST (Katoh & Standley, 2013)
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      321
Multiple spidroin families exist, corresponding to different types of
silk with various mechanical properties, but they all share conserved
non‐repetitive N/C‐terminal domains (Hayashi, Blackledge, & Lewis,
2004), and a highly complex sequence organization that is extremely
long: typically exceeding 10kbp, and is almost entirely comprised of
repetitive sequences. The multi‐giga‐base size of spider genomes
(Babb et al., 2017) and this characteristic spidroin gene structure
(Xu & Lewis, 1990) makes the determination of the full length of the
sequence challenging, as even from the PCR amplification step of
the gene there is a risk of producing chimeric artifacts. Unamplified
single molecule long nanopore sequencing is one of a very limited
number of possible approaches.
In order for an untargeted, comprehensive search for spidroin
fragments, the non‐repetitive N/C‐terminal domains as well as re‐
peat unit sequences were computationally searched from a short
read transcriptome assembly. These fragments were then extended
by an OLC of short reads until the contigs met unresolvable bifur‐
cations in the DBG. Then, the collected extended fragments were
ordered based on nanopore long reads directly obtained from mRNA
or genomic DNA to correct for base‐level accuracies, so that the
gene length and the arrangement of repeat units were guaranteed,
avoiding compressed alignment (Ricker, Qian, & Fulthorpe, 2012).
Here, we illustrate a specific example of the spidroin gene sequence
obtained by nanopore sequencer (Figure 2). Araneus ventricosus is an
orb‐weaving spider and has seven spidroin gene families with a com‐
bination of repetitive and non‐repetitive unit structures as described
above. One of the spidroin genes, the minor ampullate spidroin
(MiSp) gene, was determined by low‐coverage (5.5M reads) direct
DNA sequencing. The collection of long reads covering the full‐
length of MiSp gene sequence from numerous nanopore reads was
performed based on similarity with the MiSp gene fragments con‐
structed by short read assembly in advance. In the previous study,
it was reported that the MiSp gene sequence was over 5,000 bp in
length (Chen et al., 2012), and nanopore sequencing showed over
8.3 kbp in length. The same case of another gene (aciniform spidroin:
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322       KONO and ARAKAWA

TA B L E 1   List of bioinformatics software commonly used in the analysis of nanopore data

  Software Description Reference

Basecaller Albacore Basecaller developed by Oxford Nanopore Technologies Inc. https://nanoporetech.com

Chiron Establish end‐to‐end basecalling using a deep learning CNN+RNN+CTC (Teng et al., 2018)
structure
Metrichor Cloud‐based basecaller that cannot be run locally https://nanoporetech.com
Tombo Detection tool for modified nucleotides such as methylation https://nanoporetech.com
Polishing Nanopolish HMM‐based polishing tool using raw signal data of reads (Louis et al., 2016)
Racon Polishing tool for contigs assembled by Canu (Koren et al., 2017) with raw (Vaser, Sovic, Nagarajan, &
long reads. Sikic, 2017)
Pilon Polishing tool for contigs assembled by Canu (Koren et al., 2017) with (Walker et al., 2014)
Illumina short reads.
Assembler DBG2OLC Hybrid large genome assembler using short read contigs as anchor points (Ye, Hill, Wu, Ruan, & Ma,
for overlap graph construction with long reads 2016)
Wtdbg2 Fast long‐read assembler based on fuzzy‐Bruijn graph (FBG) that is analogy (Ruan & Li, 2019)
to de Bruijn graph but permits mismatches and gaps
RaGOO Scaffolding tool using reference‐guided contig ordering and orienting (Alonge, Soyk,
Ramakrishnan, & Wang,
2019)
MaSuRCA Hybrid genome assembler using long and short reads (Zimin et al., 2013)
Canu Canu is one of the leading Celera assembler that designed for SMRT and (Koren et al., 2017)
ONT reads. A successor of PBcR (Miller et al., 2008)
SMARTdenovo Fast and reasonably accurate OLC assembler for long reads without prior (Giordano et al., 2017)
error correction step
TULIP Efficient assembler for large and complex genome using seed extension (Jansen et al., 2017)
Falcon Long read assembler for phased diploid genome (Chin et al., 2016)
Miniasm Efficient assembler for SMRT and ONT reads without an error correction (Li, 2016)
Unicycler Assembler specially designed for hybrid assembly of small (bacterial or (Wick, Judd, Gorrie, & Holt,
viral) genomes based on SPAdes 2017)
ABruijn De novo assembler for long and noisy read using de Bruijn graph (Lin et al., 2016)
HINGE Long read OLC assembler based without pre‐assembly or read correction (Kamath, Shomorony, Xia,
step Courtade, & Tse, 2017)
SV/SNV NanoSV Breakpoint junction detection of structural variant using mapping BAM file (Cretu Stancu et al., 2017)
caller produced by LAST
Sniffles Structural variation caller using automatic filtering of false events based on (Sedlazeck et al., 2018)
coverage data
npInv Specially designed tool for non‐allelic homologous recombination (NAHR) (Shao et al., 2018)
inversions
marginCaller SNV detection program in marginAlign package https://github.com/
benedictpaten/marginAlign
Alignment minimap2 Fast mapping and alignment program for long noisy reads (Li, 2018)
minialign Fast and moderately accurate alignment tool for long reads https://github.com/ocxtal/
minialign
LAST Genome‐scale sequence comparison tool to find similar region (Kielbasa, Wan, Sato,
Horton, & Frith, 2011)
NGMLR Long read aligner with a new convex gap‐cost scoring model for SNV caller (Sedlazeck et al., 2018)
(Sniffles)
marginAlign Package to align reads to a reference genome for SNV calling (marginCaller) https://github.com/
benedictpaten/marginAlign
BWA‐MEM Leading short read mapping tool tuned to work with ONT recently https://github.com/lh3/bwa
Others NanoPipe Interactive web‐based tool for fast and easy processing and analysis of the (Shabardina et al., 2019)
ONT data
BulkVis Visualization tool for bulk FAST5 files obtained from ONT sequencing (Payne et al., 2018)
KONO and ARAKAWA
AcSp) is also shown in Figure 2. Because of the unamplified single
molecule long read sequencing of genomic DNA, not only the gene
length, but also the repetitive or exon/intron structure were clearly
uncovered.
7 | LI M ITATI O N S
The largest limitation of the nanopore sequencer is the compara‐
tively lower read accuracy when compared to short read sequenc‐
ers. Because insertions and deletions are included in the errors,
nanopore reads per se are not optimal for single nucleotide varia‐
tion (SNV) detection. Although SNV genotyping with nanopore‐se‐
quenced reads has been demonstrated, high coverage reads were
required (Ebler, Haukness, Pesout, Marschall, & Paten, 2018; Koren
et al., 2017), and HLA genotyping also has problems in that it cannot
distinguish specific alleles due to a lack of read accuracy (Jain et al.,
2018).
Improving the accuracy of nanopore sequencers depends on both
the pore chemistry and the base‐calling algorithm (Patel et al., 2018).
The latest R9.X nanopore is derived from the E. coli Curlin sigma S‐
dependent growth (CsgG) gene (Goyal et al., 2014), achieving sig‐
nificantly reduced error rate (Patel et al., 2018; Rang et al., 2018).
According to the Nanopore Community Meeting 2018 (https://
nanoporetech.com/resource-centre/videos/community-meeting), a
new protein, R10 nanopore, is due to be released in 2019, further
improving the accuracy for homopolymers. So far, R9.X nanopore
has a sharp reader that can only dominate five bases, making ac‐
curate determination of the homopolymers difficult. However, the
new chemistry has long and multiple “readers” which can dominate
more base signal, so homopolymer sequence accuracy is projected
to improve from Q34 of R9.4 to Q40 at 75× coverage.
Basecalling algorithms that convert raw ionic current signal to
nucleotide sequences based on a deep learning approach, combin‐
ing a convolutional neural network (CNN), connectionist temporal
classification, and a RNN, improved the read accuracy dramatically
(Rang et al., 2018) over the previous HMM‐based approach from
segmented raw data according to k‐mer (Boza, Brejova, & Vinar,
2017; Stoiber & Brown, 2017). As described above, many limitations
can be overcome by improved software fine‐tuned for use with
nanopore data. Many bioinformatics tools specialized or optimized
for nanopore sequencing have been released, and many more are in
active development. Table 1 lists representative software tools use‐
ful in the analysis of nanopore data.
AC K N OW L E D G M E N T S
The authors would like to thank Dr. James Fleming for his diligent
proofreading of this manuscript. This work was funded by the
ImPACT Program of Council for Science, Technology and Innovation
(Cabinet Office, Government of Japan) and in part by research funds
from the Yamagata Prefectural Government and Tsuruoka City,
Japan.
|
      323
ORCID
Nobuaki Kono  https://orcid.org/0000-0001-5960-7956
Kazuharu Arakawa  https://orcid.org/0000-0002-2893-4919
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How to cite this article: Kono N, Arakawa K. Nanopore
sequencing: Review of potential applications in functional
genomics. Develop Growth Differ. 2019;61:316–326. https://doi.
org/10.1111/dgd.12608